Combination therapy with CD4 lymphocyte depletion and mTOR inhibitors

ABSTRACT

The invention provides methods for treating a malignant neoplastic cell proliferative disorder or disease, comprising administering to a subject in need thereof an effective amount of an mTOR inhibitor and an effective amount of a CD4 lymphocyte depleting agent. Such methods find utility in the treatment of certain subsets of malignant neoplastic cell proliferative disorders or diseases, e.g. renal cell carcinoma and melanoma. The invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of an mTOR inhibitor and an effective amount of a CD4 lymphocyte depleting agent in a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International ApplicationPCT/US2011/033191, filed Apr. 20, 2011, which designated the U.S. andthat International Application was published under PCT Article 21(2) inEnglish. The present application also claims the benefit of the filingdate of U.S. Provisional Application No. 61/326,095 filed Apr. 20, 2010,the disclosures of each of which are incorporated herein by reference intheir entirety.

GOVERNMENT RIGHTS

The invention was made with government support under Grant No.K23CA120075-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods for treating malignant neoplastic cellproliferative disorders with mTOR inhibitors and CD4 lymphocytedepleting agents.

BACKGROUND

All publications cited herein are incorporated by reference in theirentirety to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The following description includesinformation that may be useful in understanding the present invention.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed invention, or that anypublication specifically or implicitly referenced is prior art.

Cancer vaccines have the potential to target tumors while sparing normaltissue. There is also renewed interest in immunotherapies formalignancies; the FDA recently approved Provenge®, which will be thefirst commercially available cancer vaccine for the treatment of a solidtumor (15). It is surprising that Provenge® has been approved foradvanced prostate cancer, which is not considered a classicimmunoresponsive disease. This suggests that cancer vaccines may beeffective for many malignancies, and it is expected that importantlesson learned while developing Provenge® will facilitate thedevelopment of vaccines for other malignancies.

The mammalian target of rapamycin, mTOR (also known as mechanistictarget of rapamycin and FK506 binding protein 12-rapamycin associatedprotein 1 (FRAP1)), is a protein which in humans is encoded by the FRAP1gene (1, 2). mTOR is a serine/threonine protein kinase involved in theregulation of protein synthesis, transcription, cell growth, cellproliferation, cell motility, and cell survival (3, 4). mTOR is apivotal regulator of cell proliferation. mTOR integrates the input fromupstream pathways, including mitogens, insulin, and growth factors (suchas IGF-1 and IGF-2) (3). In addition, mTOR senses cellular nutrient andenergy levels and cellular redox status (5). The mTOR pathway isdysregulated in many human diseases, especially certain cancers (4).

The immunosuppressive effects of mTOR inhibition are well known; mTORinhibition with rapamycin or one of the rapamycin analogs is part of thestandard regimen for immune suppression following organ transplantation.Rapamycin, the prototypic mTOR inhibitor, is a bacterial product thatcan inhibit mTOR by associating with its intracellular receptor FKBP12(6, 7). The FKBP12-rapamycin complex binds directly to theFKBP12-Rapamycin Binding (FRB) domain of mTOR (7). Rapamycin is widelyused to suppress the immune system and prevent rejection of solid organtransplants. The best characterized immunosuppressive effects ofrapamycin are based on its activities against T cells and antigenpresenting cells (APCs). In mice, rapamycin causes thymic involution(16) and inhibits T cell development (17, 18), proliferation, andmigration (19). When compared to effector T cells, regulatory T (Treg)cells are less sensitive to mTOR inhibition; therefore, the Tregpopulation becomes overrepresented (20). In addition, rapamycin maydirectly induce Treg formation; mTOR inhibition has been shown to make Tcells more sensitive to TGF-β-induced Treg-differentiation (21).Dendritic cells (DCs) have also been described as targets of mTORinhibition; mTOR inhibitors can suppress DC maturation (22) byinterfering with antigen uptake (23). mTOR treated DCs are unable tostimulate effector T cells and may even promote the differentiation ofTreg cells (23, 24). Thus, recent reports attributing immune stimulatingeffects to mTOR inhibition are surprising. These reports show thatrapamycin can enhance vaccines targeting bacterial or virus in mousemodels (9).

Renal cell carcinoma (RCC) is a classic immunoresponsive tumor. However,an effective cancer vaccine is not available for clinical use. Forpatients with metastatic RCC, the historical 3-year survival is lessthan 5% (10). Of all urologic malignancies, RCC has the highest ratio ofdisease-related deaths to incidence. The standard treatments formetastatic RCC include immune cytokines and small molecule targetedtherapies. With targeted therapies, complete responses are rare,occurring in only 1% of patients, and patients with partial responseseventually progress and succumb to the disease (11, 12). In contrast,high dose interleukin-2 (IL2) produces complete responses and durableremissions in 5-10% of patients with metastatic RCC (10, 13, 14, 15).According to certain embodiments and as disclosed herein, the inventorsshow that immune-based therapies provide treatment of the advanceddisease state of RCC and/or melanoma.

CD4 expressing lymphocytes include both helper T cells and regulatory Tcells. T helper cells are critical to mounting an adoptive immuneresponse. However, regulatory T cells (Tregs) inhibit the function ofcytotoxic T cells and normally function to limit an immune response.Therefore, the inventors evaluated CD4 depletion as a strategy forremoving Treg activity. Although only a small fraction of CD4lymphocytes are Treg cells, CD4 depletion remains an effective approachfor depleting Treg activity, and importantly, it has the potential forrapid translation to clinical use. There are humanized CD4 depletingantibodies being evaluated in clinical trials. However, there iscurrently no way to specifically target Foxp3 expressing cells inpatients. Applicants show that the combination of mTOR inhibition andCD4 depletion has a potent antitumor immune effect, capable ofinhibiting the growth of established tumors as well as hematogenousmetastasis.

SUMMARY OF THE INVENTION

The invention provides methods for treating malignant neoplastic cellproliferative disorders or diseases in subjects in need thereofcomprising administering an effective amount of an mTOR inhibitor and aneffective amount of a CD4 lymphocyte depleting agent so as to treatmalignant neoplastic cell proliferative disorders or diseases.

The invention further provides methods for inhibiting neoplastic cellproliferative disorders or diseases in subjects in need thereofcomprising administering an effective amount of an mTOR inhibitor and aneffective amount of a CD4 lymphocyte depleting agent so as to inhibitneoplastic cell proliferative disorders or diseases.

Also provided herein are methods for promoting prophylaxis of malignantneoplastic cell proliferative disorders or diseases in a subject in needthereof comprising administering an effective amount of an mTORinhibitor and an effective amount of a CD4 lymphocyte depleting agent soas to promote prophylaxis of malignant neoplastic cell proliferativedisorders or diseases.

The invention further provides pharmaceutical compositions comprising atherapeutically effective amount of an mTOR inhibitor and an effectiveamount of a CD4 lymphocyte depleting agent in a pharmaceuticallyacceptable carrier.

The invention also provides kits for treatment of malignant neoplasticcell proliferative disorders or diseases and/or inhibition of malignantneoplastic cell proliferative disorders or diseases and/or promotingprophylaxis of malignant neoplastic cell proliferative disorders ordiseases. The kits comprise an mTOR inhibitor, a CD4 lymphocytedepleting agent and instructions for use of the composition fortreatment of malignant neoplastic cell proliferative disorders ordiseases and/or inhibition of malignant neoplastic cell proliferativedisorders or diseases and/or promoting prophylaxis of malignantneoplastic cell proliferative disorders or diseases.

The claimed methods, compositions and kits find utility in the treatmentof certain subsets of malignant neoplastic cell proliferative disordersor diseases, including but not limited to renal cell carcinomas andmelanomas.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 shows that mTOR has immunosuppressive activity. (A) Tersirolimusand Rapamycin have similar activities. (B) Foxp3 positive lymphocytes(Treg cells) increase in response to mTOR inhibitors.

FIG. 2 shows that, in accordance with an embodiment of the invention,the combination of CD4 depletion and termsirolimus was an effectivetreatment for B16 tumors.

FIG. 3 shows that, in accordance with an embodiment of the invention,the combination of CD4 depletion and termsirolimus was an effectivetreatment for RENCA tumors.

FIG. 4 shows that, in accordance with an embodiment of the invention,combinational therapy produces antitumor immune memory.

FIG. 5 shows that, in accordance with an embodiment of the invention,combinational therapy produces antitumor immune memory effect thatinhibits tumor growth in a mouse model of metastatic disease.

FIG. 6 shows that, in accordance with an embodiment of the invention,combinational therapy activates T lymphocytes as measured by theIFN-gamma (IFN-γ) response.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, preventing the disease condition from developing, loweringthe chances of a patient developing the disease condition and prolonginga patient's life or life expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, brain tumor,breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastriccancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, thyroid cancer,renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer,and prostate cancer, including but not limited to androgen-dependentprostate cancer and androgen-independent prostate cancer.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

“Conditions” and “disease conditions,” as used herein may include, butare in no way limited to any form of malignant neoplastic cellproliferative disorders or diseases. Examples of such disorders includebut are not limited to cancer. Examples of cancer include, but are notlimited to, brain tumor, breast cancer, colon cancer, carcinoma, lungcancer, hepatocellular cancer, gastric cancer, pancreatic cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, thyroid cancer, renal cancer, renal cell carcinoma,melanoma, head and neck cancer, brain cancer, and prostate cancer,including but not limited to androgen-dependent prostate cancer andandrogen-independent prostate cancer.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition,prevent the pathologic condition, pursue or obtain beneficial results,or lower the chances of the individual developing the condition even ifthe treatment is ultimately unsuccessful. Those in need of treatmentinclude those already with the condition as well as those prone to havethe condition or those in whom the condition is to be prevented.

As used herein, “CD4 lymphocytes” refer to lymphocytes which expressCD4, i.e lymphocytes which are CD4+. CD4 lymphocytes may be T cellswhich express CD4.

The inventors have discovered that mTOR inhibition can enhance cancervaccines by augmenting the immune response. In transplant patients, mTORinhibitors are believed to help prevent organ rejection by increasingthe activity of regulatory T cells (Treg). Therefore, as demonstrated inthe Examples below, Applicants tested the effect of drugs that cancounter the immune suppressing effects of mTOR inhibitors and discoveredthat immune stimulation can be further enhanced by these drugs.Consequently, Applicants have combined the use of mTOR inhibitors withadditional drugs that deplete cells such as CD4 T cells (i.e. T cellswhich express CD4), thus creating a synergistic effect. According tocertain embodiments, CD4 lymphocyte (i.e. lymphocytes which express CD4)depletion is used as a strategy for removing Treg activity.

The present invention provides compositions comprising one or more mTORinhibitors and/or CD4 lymphocyte depleting agents as well as methodsthat employ these inventive mTOR inhibitors and/or CD4 lymphocytedepleting agents in in vivo and ex vivo applications where it isadvantageous to deplete the numbers of CD4 lymphocytes and/or to reduceor eliminate the activity of mTOR or a functionally-downstream molecule.

The inventors have developed a treatment for certain subsets of cancers(e.g. renal cell carcinomas and melanomas) utilizing mTOR inhibitorsand/or CD4 lymphocyte depleting agents. The inventors have determined asynergist effect of mTOR inhibitors with CD4 lymphocyte depleting agentson certain types of cancers (e.g. renal cell carcinomas and melanomas).According to particular aspects, the invention encompassespharmaceutical compositions comprising mTOR inhibitors and/or CD4lymphocyte depleting agents. According to further aspects, the inventorshave combined the use of mTOR inhibitors with additional drugs thatdeplete cells such as CD4 T cells, and as such have created asynergistic effect for the treatment of malignant neoplastic cellproliferative disorders or diseases, e.g. renal cell carcinomas andmelanomas. According to certain aspects, mTOR inhibitors and CD4lymphocyte depleting agents are used in combination with otherpharmaceuticals, including but not limited to cancer vaccines.

Therapeutic Methods of the Invention

The invention provides methods for treating malignant neoplastic cellproliferative disorders or diseases in subjects in need thereof. Themethod comprises providing a composition comprising an mTOR inhibitorand a CD4 lymphocyte depleting agent and administering a therapeuticallyeffective amount of the composition to the subject to treat a malignantneoplastic cell proliferative disorder or disease. The composition mayfurther comprise other pharmaceuticals, including but not limited tocancer vaccines. In one embodiment, the mTOR inhibitor is a macrolidecompound including but not limited to Temsirolimus (CCI-779) or apharmaceutical equivalent, analog, derivative or a salt thereof,Evirolimus (RAD001) or a pharmaceutical equivalent, analog, derivativeor a salt thereof, and/or Rapamycin or a pharmaceutical equivalent,analog, derivative or a salt thereof. In another embodiment, an agentfor depleting CD4 lymphocytes is an antibody, for example, a humanizedanti-CD4 antibody such as zanolimumab. In some embodiments, malignantneoplastic cell proliferative disorder or disease is renal cellcarcinoma or melanoma.

The invention further provides methods for inhibiting malignantneoplastic cell proliferative disorders or diseases in subjects in needthereof. The method comprises providing a composition comprising an mTORinhibitor and a CD4 lymphocyte depleting agent, and administering atherapeutically effective amount of the composition to the subject toinhibit a malignant neoplastic cell proliferative disorder or disease.The composition may further comprise other pharmaceuticals, includingbut not limited to cancer vaccines. In one embodiment, the mTORinhibitor is a macrolide compound including but not limited toTemsirolimus (CCI-779) or a pharmaceutical equivalent, analog,derivative or a salt thereof, Evirolimus (RAD001) or a pharmaceuticalequivalent, analog, derivative or a salt thereof, and/or Rapamycin or apharmaceutical equivalent, analog, derivative or a salt thereof. Inanother embodiment, an agent for depleting CD4 lymphocytes is anantibody, for example, a humanized anti-CD4 antibody such aszanolimumab. In some embodiments, malignant neoplastic cellproliferative disorder or disease is renal cell carcinoma or melanoma.

The invention further provides methods for reducing the tumor sizeassociated malignant neoplastic cell proliferative disorders or diseasesin subjects in need thereof. The method comprises providing acomposition comprising an mTOR inhibitor and a CD4 lymphocyte depletingagent, and administering a therapeutically effective amount of thecomposition to the subject to reduce tumor size associated withmalignant neoplastic cell proliferative disorder or disease. Thecomposition may further comprise other pharmaceuticals, including butnot limited to cancer vaccines. In one embodiment, the mTOR inhibitor isa macrolide compound including but not limited to Temsirolimus (CCI-779)or a pharmaceutical equivalent, analog, derivative or a salt thereof,Evirolimus (RAD001) or a pharmaceutical equivalent, analog, derivativeor a salt thereof, and/or Rapamycin or a pharmaceutical equivalent,analog, derivative or a salt thereof. In another embodiment, an agentfor depleting CD4 lymphocytes is an antibody, for example, a humanizedanti-CD4 antibody such as zanolimumab. In some embodiments, malignantneoplastic cell proliferative disorder or disease is renal cellcarcinoma or melanoma.

The invention also provides methods for promoting prophylaxis ofmalignant neoplastic cell proliferative disorders or diseases insubjects in need thereof. The method comprises providing a compositioncomprising an mTOR inhibitor and a CD4 lymphocyte depleting agent, andadministering a therapeutically effective amount of the composition tothe subject to promote prophylaxis of malignant neoplastic cellproliferative disorder or disease. The composition may further compriseother pharmaceuticals, including but not limited to cancer vaccines. Inone embodiment, the mTOR inhibitor is a macrolide compound including butnot limited to Temsirolimus (CCI-779) or a pharmaceutical equivalent,analog, derivative or a salt thereof, Evirolimus (RAD001) or apharmaceutical equivalent, analog, derivative or a salt thereof, and/orRapamycin or a pharmaceutical equivalent, analog, derivative or a saltthereof. In another embodiment, an agent for depleting CD4 lymphocytesis an antibody, for example, a humanized anti-CD4 antibody such aszanolimumab. In some embodiments, malignant neoplastic cellproliferative disorder or disease is renal cell carcinoma or melanoma.

In some embodiments of the invention, the mTOR inhibitor may be any oneor more of a small molecule, a peptide, an antibody or a fragmentthereof, a nucleic acid molecule and/or a macrolide compound. In anembodiment, the antibody specifically binds mTOR so as to inhibit mTOR.The antibody may be any one or more of a monoclonal antibody or fragmentthereof, a polyclonal antibody or a fragment thereof, a chimericantibody, a humanized antibody, a human antibody or a single chainantibody. These antibodies can be from any source, e.g., rat, dog, cat,pig, horse, mouse or human. Fragments of antibodies may be any one ormore of Fab, F(ab′)2, Fv fragments or fusion proteins.

In a preferred embodiment of the invention, the mTOR inhibitor is amacrolide compound. Examples of macrolide compounds that may be usedwith the claimed invention include but are not limited to Temsirolimus(CCI-779) or a pharmaceutical equivalent, analog, derivative or a saltthereof, Evirolimus (RAD001) or a pharmaceutical equivalent, analog,derivative or a salt thereof, and/or Rapamycin or a pharmaceuticalequivalent, analog, derivative or a salt thereof.

In a further embodiment of the invention, the CD4 lymphocyte depletingagent may be any one or more of small molecule, a peptide, an antibodyor a fragment thereof, a nucleic acid molecule and/or a macrolidecompound. In an embodiment, the antibody specifically binds CD4 onCD4-expressing T cells such as regulatory T cells (Treg cells). Theantibody may be any one or more of a monoclonal antibody or fragmentthereof, a polyclonal antibody or a fragment thereof, a chimericantibody, a humanized antibody, a human antibody or a single chainantibody. These antibodies can be from any source, e.g., rat, dog, cat,pig, horse, mouse or human. Fragments of antibodies may be any one ormore of Fab, F(ab′)2, Fv fragments or fusion proteins.

In a preferred embodiment of the invention, the CD4 lymphocyte depletingagent is an antibody such as a humanized anti-CD4 antibody or a fragmentthereof. In the most preferred embodiment, the CD4 lymphocyte depletingantibody is zanolimumab.

In some embodiments of the invention, the mTOR inhibitor and the CD4lymphocyte depleting agent are administered concurrently. In additionalembodiments, the mTOR inhibitor and the CD4 lymphocyte depleting agentare administered sequentially. In further embodiments, the compositioncomprising mTOR inhibitor and the CD4 lymphocyte depleting agent areadministered with food or without food. According to certain aspects,mTOR inhibitors and CD4 lymphocyte depleting agents may be used incombination with other pharmaceuticals, including but not limited tocancer vaccines.

As described above, methods for treating malignant neoplastic cellproliferative disorders or diseases, inhibiting malignant neoplasticcell proliferative disorders or diseases, reducing tumor size associatedwith malignant neoplastic cell proliferative disorders or diseases andpromoting prophylaxis of malignant neoplastic cell proliferativedisorders or diseases comprises providing and administering to thesubject in need thereof, a composition comprising an mTOR inhibitor anda CD4 lymphocyte depleting agent. In one embodiment, the composition foruse with the claimed methods comprises the mTOR inhibitor Temsirolimus(CCI-779) or a pharmaceutical equivalent, analog, derivative or a saltthereof and a humanized anti-CD4 antibody (such as zanolimumab) as theCD4 lymphocyte depleting agent. The mTOR inhibitor (for exampleTemsirolimus (CCI-779) or a pharmaceutical equivalent, analog,derivative or a salt thereof) and the CD4 lymphocyte depleting agent(for example zanolimumab) may be administered concurrently orsequentially. Additionally, the composition comprising the mTORinhibitor Temsirolimus (CCI-779) or a pharmaceutical equivalent, analog,derivative or a salt thereof and a humanized anti-CD4 antibody (such aszanolimumab) as the CD4 lymphocyte depleting agent may further comprisea cancer vaccine.

In another embodiment, the composition for use with the claimed methodscomprises the mTOR inhibitor Evirolimus (RAD001) or a pharmaceuticalequivalent, analog, derivative or a salt thereof and humanized anti-CD4antibody (such as zanolimumab) as the CD4 lymphocyte depleting agent.The mTOR inhibitor (for example Evirolimus (RAD001) or a pharmaceuticalequivalent, analog, derivative or a salt thereof) and the CD4 lymphocytedepleting agent (for example zanolimumab) may be administeredconcurrently or sequentially. Additionally, the composition comprisingthe mTOR inhibitor Evirolimus (RAD001) or a pharmaceutical equivalent,analog, derivative or a salt thereof and humanized anti-CD4 antibody(such as zanolimumab) as the CD4 lymphocyte depleting agent may furthercomprise a cancer vaccine.

In a further embodiment, the composition for use with the claimedmethods comprises the mTOR inhibitor is Rapamycin or a pharmaceuticalequivalent, analog, derivative or a salt thereof and humanized anti-CD4antibody (such as zanolimumab) as the CD4 lymphocyte depleting agent.The mTOR inhibitor (for example Rapamycin or a pharmaceuticalequivalent, analog, derivative or a salt thereof) and the CD4 lymphocytedepleting agent (for example zanolimumab) may be administeredconcurrently or sequentially. Additionally, the composition comprisingthe mTOR inhibitor is Rapamycin or a pharmaceutical equivalent, analog,derivative or a salt thereof and humanized anti-CD4 antibody (such aszanolimumab) as the CD4 lymphocyte depleting agent may further comprisea cancer vaccine.

The subjects treated by the present invention include mammaliansubjects, including, human, monkey, ape, dog, cat, cow, horse, goat,pig, rabbit, mouse and rat.

Various methods may be utilized to administer the composition of theclaimed methods, including but not limited to aerosol, nasal, oral,transmucosal, transdermal, parenteral, implantable pump, continuousinfusion, topical application, capsules and/or injections.

Dosages of the Invention

In some embodiments of the invention, the effective amounts of the mTORinhibitors in the composition for use with the claimed methods, in thepharmaceutical compositions and/or in the claimed kits may be in therange of about 0.1-0.5 mg/day, 0.5-1.0 mg/day, 1.0-1.5 mg/day, 1.5-2mg/day, 2.0-2.5 mg/day, 2.5-5 mg/day, 5-10 mg/day, 10-15 mg/day, 15-20mg/day, 20-25 mg/day, 25-30 mg/day, 30-35 mg/day, 35-40 mg/day, 40-45mg/day, 45-50 mg/day, 50-55 mg/day, 55-60 mg/day, 60-65 mg/day, 65-70mg/day, 70-75 mg/day, 75-80 mg/day, 80-85 mg/day, 85-90 mg/day, 90-95mg/day, 95-100 mg/day, 0.75-10 mg/day or 2-10 mg/day. In someembodiments of the invention, the mTOR inhibitors are Temsirolimus(CCI-779) or a pharmaceutical equivalent, analog, derivative or a saltthereof, Evirolimus (RAD001) or a pharmaceutical equivalent, analog,derivative or a salt thereof, and/or Rapamycin or a pharmaceuticalequivalent, analog, derivative or a salt thereof.

In alternate embodiments, the effective amounts of the mTOR inhibitorsin the composition for use with the claimed methods, in thepharmaceutical compositions and/or in the claimed kits may be in therange of about 1-5 mg/week, 5-10 mg/week, 10-15 mg/week, 15-20 mg/week,20-25 mg/week, 25-30 mg/week, 30-35 mg/week, 35-40 mg/week, 40-45mg/week, 45-50 mg/week, 50-55 mg/week, 55-60 mg/week, 60-65 mg/week,65-70 mg/week, 70-75 mg/day, 75-80 mg/mg, 80-85 mg/mg, 85-90 mg/week,90-95 mg/week or 95-100 mg/week. In some embodiments of the invention,the mTOR inhibitors are Temsirolimus (CCI-779) or a pharmaceuticalequivalent, analog, derivative or a salt thereof, Evirolimus (RAD001) ora pharmaceutical equivalent, analog, derivative or a salt thereof,and/or Rapamycin or a pharmaceutical equivalent, analog, derivative or asalt thereof.

In some preferred embodiments, Temsirolimus is administered at a dose of25 mg over 30-60 minutes per week, Evirolimus is administered at a doseof 0.75-10 mg per day and/or Rapamycin is administered at a dose of 2-10mg per day.

In some embodiments of the invention, the effective amounts of the CD4lymphocyte depleting agent in the composition for use with the claimedmethods, in the pharmaceutical compositions and/or in the claimed kitsmay be in the range of about 100-200 mg/day, 200-300 mg/day, 300-400mg/day, 400-500 mg/day, 500-600 mg/day, 600-700 mg/day, 700-800 mg/day,800-900 mg/day, 900-1000 mg/day, 1000-1100 mg/day, 1100-1200 mg/day,1200-1300 mg/day, 1300-1400 mg/day, 1400-1500 mg/day, 1500-1600 mg/day,1600-1700 mg/day, 1700-1800 mg/day, 1800-1900 mg/day or 1900-2000mg/day. In a preferred embodiment of the invention, the CD4 lymphocytedepleting agent is a humanized anti-CD4 antibody (for exampleZanolimumab dose).

In other embodiments of the invention, the effective amounts of the CD4lymphocyte depleting agent in the composition for use with the claimedmethods, in the pharmaceutical compositions and/or in the claimed kitsmay be in the range of about 100-200 mg/week, 200-300 mg/week, 300-400mg/week, 400-500 mg/week, 500-600 mg/week, 600-700 mg/week, 700-800mg/week, 800-900 mg/week, 900-1000 mg/week, 1000-1100 mg/week, 1100-1200mg/week, 1200-1300 mg/week, 1300-1400 mg/week, 1400-1500 mg/week,1500-1600 mg/week, 1600-1700 mg/week, 1700-1800 mg/week, 1800-1900mg/week or 1900-2000 mg/week. In a preferred embodiment of theinvention, the CD4 lymphocyte depleting agent is a humanized anti-CD4antibody (for example Zanolimumab dose). Zanolimumab may be administeredat a dose of 980 mg per week.

In an embodiment of the claimed methods of the invention, the mTORinhibitor and the CD4 lymphocyte depleting agent may be administeredsimultaneously at the aforementioned dosages using the appropriate modesof administration, for instance, the modes of administration recommendedby the manufacturer for each of the mTOR inhibitor and the CD4lymphocyte depleting agent.

Alternately, the mTOR inhibitor and the CD4 lymphocyte depleting agentmay be administered sequentially at the aforementioned dosages. Forexample, the mTOR inhibitors (for example Temsirolimus, Evirolimus orRapamycin) may be administered, for example, daily at the aforementioneddosages and the CD4 lymphocyte depleting agent (for example a humanizedanti-CD4 antibody) may be administered for example, daily, weekly,biweekly, every fortnight and/or monthly at the aforementioned dosages.Alternately, the mTOR inhibitors (for example Temsirolimus, Evirolimusor Rapamycin) may be administered, for example, daily, weekly, biweekly,every fortnight and/or monthly, at the aforementioned dosages and theCD4 lymphocyte depleting agent (for example a humanized anti-CD4antibody) may be administered for example, daily, at the aforementioneddosages. Further, each of the mTOR inhibitor (for example Temsirolimus,Evirolimus or Rapamycin) and the CD4 lymphocyte depleting agent (forexample a humanized anti-CD4 antibody) may be administered daily,weekly, biweekly, every fortnight and/or monthly, wherein the mTORinhibitor is administered at the aforementioned dosages on a daydifferent than the day on which the CD4 lymphocyte depleting agent isadministered at the aforementioned dosages.

The cancer vaccine dose would depend on the vaccine being used. Theeffective dose of the cancer vaccine may be determined by one skilled inthe art (such as the physician) or it may be administered per themanufacturers' recommendation. In one embodiment, the first dose of thecancer vaccine is administered on day 0 and the second dose isadministered on day 7. mTOR inhibitors may be administered on days 2-32,at the aforementioned dosages. Further, 2-3 weekly doses of anti-CD4depleting agent may be administered starting on day 10, at theaforementioned dosages. For example, if a heat shock protein vaccine isused, a heat shock protein (for example, hsp110 or grp170) may becomplexed with a tumor antigen (such as gp100) and subsequentlyadministered. In an embodiment, for a melanoma vaccine, a complex ofhsp110 and gp100 at 2.5 mg/kg, may be administered intradermally.

Typical dosages of an effective amount of mTOR inhibitors (for exampleTemsirolimus (CCI-779) or a pharmaceutical equivalent, analog,derivative or a salt thereof, Evirolimus (RAD001) or a pharmaceuticalequivalent, analog, derivative or a salt thereof, and/or Rapamycin or apharmaceutical equivalent, analog, derivative or a salt thereof) or CD4lymphocyte depleting agent can be in the ranges recommended by themanufacturer where known therapeutic compounds are used, and also asindicated to the skilled artisan by the in vitro responses or responsesin animal models.

For example, the FDA approved dosage for Temsirolimus is 25 mgadministered intravenously over 30-60 min every week, for Evirolimus is0.75 mg-10 mg per day administered orally, for Rapamycin is about 2-10mg per day administered orally and for Zanolimumab is about 980 mg perweek administered intravenously. The same or similar dosing can be usedin accordance with various embodiments of the present invention, or analternate dosage may be used in connection with alternate embodiments ofthe invention. The actual dosage can depend upon the judgment of thephysician, the condition of the patient, and the effectiveness of thetherapeutic method based, for example, on the in vitro responsiveness ofrelevant cultured cells or histocultured tissue sample, or the responsesobserved in the appropriate animal models.

Pharmaceutical Compositions of the Invention

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of a mTOR inhibitor (for exampleTemsirolimus (CCI-779) or a pharmaceutical equivalent, analog,derivative or a salt thereof, Evirolimus (RAD001) or a pharmaceuticalequivalent, analog, derivative or a salt thereof, and/or Rapamycin or apharmaceutical equivalent, analog, derivative or a salt thereof) and aCD4 lymphocyte depleting agent (for example humanized anti-CD4 antibodysuch as Zanolimumab). “Pharmaceutically acceptable excipient” means anexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and desirable, and includes excipientsthat are acceptable for veterinary use as well as for humanpharmaceutical use. Such excipients may be solid, liquid, semisolid, or,in the case of an aerosol composition, gaseous. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral, topicalor local for therapeutic treatment. Preferably, the compositions areadministered orally or parenterally, i.e., intravenously,intraperitoneally, intradermally, or intramuscularly.

mTOR inhibitors and/or CD4 lymphocyte depleting agents useful in thetreatment of disease in mammals will often be prepared substantiallyfree of naturally-occurring immunoglobulins or other biologicalmolecules. Preferred mTOR inhibitors and/or CD4 lymphocyte depletingagents will also exhibit minimal toxicity when administered to a mammal.

The compositions of the invention may be sterilized by conventional,well-known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may containpharmaceutically-acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, and stabilizers (e.g., 1-20% maltose, etc.).

According to certain aspects, mTOR inhibitors may include, but are notlimited to, rapamycin, Temsirolimus, (CCI-779) and Evirolimus (RAD001).According to certain aspects, CD4 lymphocyte depleting agents include,but are not limited to, humanized anti-CD4 antibodies such aszanolimumab.

Kits of the Invention

The present invention is also directed to articles of manufacture andkits containing mTOR inhibitors and CD4 lymphocyte depleting agents fortherapeutic methods described above. mTOR inhibitors include but are notlimited to mTOR inhibitor (for example Temsirolimus (CCI-779) or apharmaceutical equivalent, analog, derivative or a salt thereof,Evirolimus (RAD001) or a pharmaceutical equivalent, analog, derivativeor a salt thereof, and/or Rapamycin or a pharmaceutical equivalent,analog, derivative or a salt thereof). CD4 lymphocyte depleting agentsinclude but are not limited to humanized anti-CD4 antibody, such asZanolimumab.

In one embodiment, the kit is configured particularly for the purpose oftreating mammalian subjects. In another embodiment, the kit isconfigured particularly for the purpose of treating human subjects. Infurther embodiments, the kit is configured for veterinary applications,treating subjects such as, but not limited to, farm animals, domesticanimals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to affect a desired outcome.Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia as will be readily recognized bythose of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

EXAMPLES

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Example 1 mTOR has Immunosuppressive Activity

As has been well established, mTOR inhibitors have immune suppressingactivity. For the [3H] thymidine incorporation assay, lymph nodes wereharvested from naïve C57 BL/6 or Pmel-1 mouse. 3×10⁵ cells/well werecultured in 96-well plates and stimulated, with or without mTORinhibitors, for 72 hours. C57 BL/6 lymphocytes were stimulated withanti-CD3 and anti-CD28 mAb, and Pmel-1 lymphocytes were stimulated withgp100 peptide. DNA synthesis was determined by incubation for 16 h with1 μCi [3H]thymidine (Amersham Biosciences, Piscataway, N.J., USA). Asshown in FIG. 1A the two mTOR inhibitors, namely temsirolimus andrapamycin have nearly identical activities.

Further, in vivo effect of temsirolimus on CD4+FoxP3+ lymphocytes wasassessed. B6 mice (5 mice per group) were treated daily withtemsirolimus for 24 days, 2 doses of vaccine (complex of hsp110 andgp100), or both. FoxP3 staining was quantified with flow cytometry whilegating on CD4. As shown in FIG. 1B, Foxp3 positive lymphocytes(regulatory T cells) are increased by mTOR inhibition. Data show meanand standard error of the mean (s.e.m). Representative results are shownfrom at least 2 independent experiments. Based on these data, inventorsadapted the strategy of CD4 depletion as a strategy for removing theseregulatory T cells (Tregs).

Example 2 The Combination of CD4 Depletion and Termsirolimus was anEffective Treatment for B16 Tumors

In this Example, the inventors tested their combinational therapystrategy, wherein mTOR inhibitors were combined with a strategy todeplete regulatory T cells (Treg cells) in mouse tumor model of B16melanoma. Mice with established B16 melanoma tumors, which are veryimmunoresistent tumors, were treated with the combination oftemsirolimus and CD4 depleting antibodies.

Ten days after subcutaneous implantation of tumor cells B6 mice weretreated with PBS (control), temsirolimus, CD4 depleting antibody(intraperitoneal injection of 200 μg of GK1.5 mAb, administered everyother day for 6 days, starting at day 4), or both (temsirolimus and CD4depleting antibody). The tumor growth was monitored. Each line in FIG. 2represents mean tumor growth and standard error of the mean (s.e.m), infive animals. P-values are for repeated measures ANOVA. As shown in FIG.2, this combination was effective in slowing the growth of B16 tumors.

Example 3 Combination of CD4 Lymphocyte Depletion and Temsirolimus wasan Effective Treatment for RENCA Tumors

In this Example, the inventors tested their combinational therapystrategy, wherein mTOR inhibitors were combined with a strategy todeplete regulatory T cells (Treg cells) in mouse tumor model of RENCAtumors. RENCA is a murine renal cancer cell and model.

Ten days after subcutaneous implantation of RENCA tumor cells, Balb/cmice were treated with PBS (control), temsirolimus, CD4 depletingantibody (intraperitoneal injection of 200 μg of GK1.5 mAb, administeredevery other day for 6 days, starting at day 4), or both (temsirolimusand CD4 depleting antibody). The tumor growth was monitored. Each linein FIG. 3 represents mean tumor growth and standard error of the mean(s.e.m) in five animals. P-values are for repeated measures ANOVA. Asshown in FIG. 3, this combination was effective in curing three of fivemice with established tumors, even though a specific tumor vaccine wasnot included.

Example 4 Combinational Therapy Strategy Produces Antitumor ImmuneMemory

Ten days after subcutaneous implantation of 2×10⁵ tumor cells, Balb/cmice were treated with temsirolimus (30 ug/mouse daily on days 14 to 34,intraperitoneally) or temsirolimus plus CD4 depleting antibody(intraperitoneal injection of 200 μg of GK1.5 mAb, administered at day 6and 10). Mice were re-challenged with 2×10⁵ RENCA tumor cells on day 47.Naïve mouse group was also challenged with RENCA and served as acontrol. Tumor size was measured 11 and 18 days after the re-challenge.The tumor grew at the same rate in untreated mice and mice treated withtemsirolimus alone. However, in the mice treated with both temsirolimusand CD4 depletion the tumor did not grow (FIG. 4). Since the mice werenot being actively treated at the time of tumor re-challenge, directantitumor effects of the therapy cannot account for the effects on tumorgrowth. These data suggest that indirect immune effects persist aftertreatment and account for the observed inhibition of tumor growth.

Example 5 Combinational Therapy Produces Antitumor Immune Memory Effectthat Inhibits Tumor Growth in a Mouse Model of Metastatic Disease

RENCA tumor cells (2×10⁵ cells) were injected subcutaneously into theflank of Balb/c mice, CD4 depleting antibody was administered twice ondays 6 and 10, and temsirolimus (15 ug/mouse) was administered dailyfrom days 14 to day 34. On day 91, mice were rechallenged with RENCAtumor cells (2×10⁵) injected intravenously. The mouse lungs wereexamined for tumor growth 30 days after the rechallenge (FIG. 5). Thecombinational therapy inhibited the establishment and growth of RENCAtumors in the lung.

Example 6 Combinational Therapy Activates Cytotoxic T Lymphocytes

RENCA tumor cells expressing CA9 were injected subcutaneously into theflank of Balb/c mice. Mice were treated as described in Example 3. Lymphnodes were harvested 35 days after tumor implantation. Lymphocytes wereactivated in vitro with CA9 protein (10 μg/ml) at 37° C. for 48 h, thenwashed and intracellular stained with IFN-γ. Flow cytometry wasperformed using the FACScan to analyze IFN-gamma (IFNγ, IFN-γ)expression, while gating on CD8. As shown in FIG. 6, combination therapyactivates cytotoxic T lymphocytes as measured by the IFN-gamma response.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects. It willbe understood by those within the art that, in general, terms usedherein are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

REFERENCES

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What is claimed is:
 1. A method of treating a carcinoma or melanoma in asubject in need thereof, wherein the subject has been diagnosed with thecarcinoma or melanoma, comprising: (i) providing a compositionconsisting essentially of an mTOR inhibitor and an anti-CD4 antibody oran antigen-binding fragment thereof, wherein the mTOR inhibitor isRapamycin or a pharmaceutical equivalent, analog, derivative or a saltthereof; and (ii) administering a therapeutically effective amount ofthe composition to the subject so as to treat the carcinoma or melanoma,thereby treating the carcinoma or melanoma.
 2. A method of inhibiting acarcinoma or melanoma in a subject in need thereof, comprising: (i)providing a composition consisting essentially of an mTOR inhibitor andan anti-CD4 antibody or an antigen-binding fragment thereof, wherein themTOR inhibitor is Rapamycin or a pharmaceutical equivalent, analog,derivative or a salt thereof; and (ii) administering a therapeuticallyeffective amount of the composition to the subject to inhibit thecarcinoma or melanoma, thereby inhibiting the carcinoma or melanoma. 3.The method of claim 1 or 2, wherein the mTOR inhibitor is selected fromthe group consisting of (i) temsirolimus (CCI-779) or a pharmaceuticalequivalent, analog, derivative or a salt thereof, and (ii) Evirolimus(RAD001) or a pharmaceutical equivalent, analog, derivative or a saltthereof.
 4. The method of claim 1 or 2, wherein the anti-CD4 antibody isa monoclonal antibody or an antigen-binding fragment thereof, apolyclonal antibody or an antigen-binding fragment thereof, a chimericantibody, a humanized antibody, a human antibody or a single chainantibody.
 5. The method of claim 4, wherein the anti-CD4 antibody is ahumanized anti-CD4 antibody.
 6. The method of claim 5, wherein theanti-CD4 antibody is zanolimumab.
 7. The method of claim 1 or 2, whereinthe composition consists essentially of a humanized anti-CD4 antibodyand temsirolimus or a pharmaceutical equivalent, analog, derivative or asalt thereof.
 8. The method of claim 1 or 2, wherein the carcinoma is arenal cell carcinoma.
 9. The method of claim 1 or 2, wherein theeffective amount of the mTOR inhibitor is 0.1-0.5 mg/day, 0.5-1.0mg/day, 1.0-1.5 mg/day, 1.5-2 mg/day, 2.0-2.5 mg/day, 2.5-5 mg/day, 5-10mg/day, 10-15 mg/day, 15-20 mg/day, 20-25 mg/day, 25-30 mg/day, 30-35mg/day, 35-40 mg/day, 40-45 mg/day, 45-50 mg/day, 50-55 mg/day, 55-60mg/day, 60-65 mg/day, 65-70 mg/day, 70-75 mg/day, 75-80 mg/day, 80-85mg/day, 85-90 mg/day, 90-95 mg/day or 95-100 mg/day.
 10. The method ofclaim 1 or 2, wherein the effective amount of the anti-CD4 antibody oran antigen-binding fragment thereof is 100-200 mg/day, 200-300 mg/day,300-400 mg/day, 400-500 mg/day, 500-600 mg/day, 600-700 mg/day, 700-800mg/day, 800-900 mg/day, 900-1000 mg/day, 1000-1100 mg/day, 1100-1200mg/day, 1200-1300 mg/day, 1300-1400 mg/day, 1400-1500 mg/day, 1500-1600mg/day, 1600-1700 mg/day, 1700-1800 mg/day, 1800-1900 mg/day or1900-2000 mg/day.
 11. The method of claim 1 or 2, wherein thecomposition is administered intravenously, intramuscularly,intraperitoneally, orally or via inhalation.